Aslamo alikom/ Greetings everyone,
I'm conducting a western blot experiment (8% SDS gel) and I wanna test 2 proteins, one is 95 KDa, and another is 35KDa. The antibodies I've for both are mouse Abs used at 1:1000 dilution and I use secondary HRP-conjugated at 1:2000 dilutions.
Ideally, I use to test them sequentially, but I'm wondering if it's possible to add the 2 antibodies to the incubation buffer (BSA/milk) and test for both in one go?
I would highly appreciate an answer for this. Also, if someone has done it before, I would appreciate the feedback/tips if any.
Thank you
You can do this. Make sure you get a clean, specific band for each before combining them, since the backgrounds from each will add up.
Dear Mariam,
It is advisable not to mix two primary antibodies even if they have requirement for same secondary antibody. Cutting the blot based on the molecular weight is one approach and the second one is to follow sequential incubation.
Good luck with your blots.
Best,
Sheethal Galande
PhD Fellow
AHF
AIG Hospitals
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